This proposal will investigate virulence mechanisms of cytoadherence/sequestration using Babesia bovis as a natural host model system. Virulent B. bovis infection is associated with development of severe clinical disease and neurological manifestations similar to those observed in human patients with severe malaria caused by virulent strains of P. falciparum. The proposed unique model enables in vivo study of virulence mechanisms using defined and reproducible virulence phenotypes. In this model, the role of cytoadhesion/sequestration in determining virulence and whether specific gene products mediate these phenotypes can be determined. Published and preliminary data from three virulent and derived attenuated B. bovis pairs strongly supports the role of VESA1 proteins in virulence, and the overall hypothesis that differential VESA1 protein expression alone may be responsible for mediating virulence. Clonal lines of virulent and attenuated strain pairs will be used and their differential phenotypes confirmed. The specific VESA1 surface protein dimer associated with sequestration and expressed by virulent versus attenuated strains both in vitro and in vivo will be identified using monoclonal antibodies. Direct demonstration that a specific VESA1 dimer is associated with sequestration and virulence will be determined by complementation and expression of cytoadhesion and virulence associated ves1 gene(s) in the attenuated T2Bo clonal line, followed by an in vivo study to demonstrate acquisition of the virulence phenotype. If the hypothesis is correct, and if similar mechanisms are used in other pathogens, molecular targets for intervention to prevent severe clinical manifestations and death associated with hemoparasite infection can be further examined. Future directions will be to determine specific domain(s) of VESA1 proteins required for virulence, determine whether these domains are present in other VESA1 proteins and if so whether these proteins are also associated with virulence. Once this is established, the design and testing of virulence directed interventions such as peptide analogues or vaccines to reduce the morbidity and mortality associated with hemoparasite infection in animals and humans will be possible.